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Identification of a BRCA1-mRNA splicing complex required for efficient DNA repair and maintenance of genomic stability.

Savage KI, Gorski JJ, Barros EM, Irwin GW, Manti L, Powell AJ, Pellagatti A, Lukashchuk N, McCance DJ, McCluggage WG, Schettino G, Salto-Tellez M, Boultwood J, Richard DJ, McDade SS, Harkin DP - Mol. Cell (2014)

Bottom Line: Mutations within BRCA1 predispose carriers to a high risk of breast and ovarian cancers.Here, we report the identification of a DNA damage-induced BRCA1 protein complex containing BCLAF1 and other key components of the mRNA-splicing machinery.Interestingly, mutations in a number of proteins found within this complex have been identified in numerous cancer types.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK. Electronic address: k.savage@qub.ac.uk.

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BRCA1/BCLAF1-Mediated mRNA Splicing Is Required for Maintenance of ATRIP, BACH1, and EXO1 Protein Expression(A) Representative western blots demonstrating that depletion of either BRCA1 or BCLAF1 results in downregulated expression of ATRIP, BACH1, and EXO1 proteins in response to DNA damage.(B) Representative western blot demonstrating DNA damage-dependent accumulation of ATRIP, BACH1, and EXO1 proteins over time following inhibition of proteosomal mediated protein degradation with MG132 (10 μM). Cells were mock treated or treated with etoposide (1 μM) for 30 min prior to MG132 treatment.(C) Representative western blots demonstrating DNA damage-dependent increased protein turnover in control and BRCA1- or BCLAF1-depleted cells following inhibition of protein translation with Cyclohexamide (10 μg/mL). Cells were mock treated or treated with etoposide (1 μM) for 30 min prior to Cyclohexamide treatment.(D) Representative western blots demonstrating BRCA1 and BCLAF depletion in cells used for experiments shown in (C).(E–G) Quantification of ATRIP, BACH1, and EXO1 protein levels shown in (C). Image densitometry values were normalized to 0 hr and decay curves fitted and used to calculate protein half-lives.
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fig6: BRCA1/BCLAF1-Mediated mRNA Splicing Is Required for Maintenance of ATRIP, BACH1, and EXO1 Protein Expression(A) Representative western blots demonstrating that depletion of either BRCA1 or BCLAF1 results in downregulated expression of ATRIP, BACH1, and EXO1 proteins in response to DNA damage.(B) Representative western blot demonstrating DNA damage-dependent accumulation of ATRIP, BACH1, and EXO1 proteins over time following inhibition of proteosomal mediated protein degradation with MG132 (10 μM). Cells were mock treated or treated with etoposide (1 μM) for 30 min prior to MG132 treatment.(C) Representative western blots demonstrating DNA damage-dependent increased protein turnover in control and BRCA1- or BCLAF1-depleted cells following inhibition of protein translation with Cyclohexamide (10 μg/mL). Cells were mock treated or treated with etoposide (1 μM) for 30 min prior to Cyclohexamide treatment.(D) Representative western blots demonstrating BRCA1 and BCLAF depletion in cells used for experiments shown in (C).(E–G) Quantification of ATRIP, BACH1, and EXO1 protein levels shown in (C). Image densitometry values were normalized to 0 hr and decay curves fitted and used to calculate protein half-lives.

Mentions: As BRCA1 and BCLAF1 were required for efficient splicing and stability of spliced ATRIP, BACH1, and EXO1 transcripts following DNA damage, we next assessed the effect of BRCA1 or BCLAF1 depletion on ATRIP, BACH1, and EXO1 protein expression. In keeping with the reduced expression of ATRIP, BACH1, and EXO1 spliced transcripts, we observed substantially reduced expression of all three proteins in BRCA1- and BCLAF1-depleted cells following DNA damage (Figure 6A). However, to our surprise, in control cells we did not observe increased levels of ATRIP, BACH1, and EXO1 protein levels following DNA damage that would be expected, given the increased expression of spliced transcript observed. This suggests that either ATRIP, BACH1, and EXO1 translation is attenuated following DNA damage or or that turnover of these proteins may be increased and that BRCA1/BCLAF1-regulated cotranscriptional splicing serves to maintain the expression of these proteins in response to DNA damage.


Identification of a BRCA1-mRNA splicing complex required for efficient DNA repair and maintenance of genomic stability.

Savage KI, Gorski JJ, Barros EM, Irwin GW, Manti L, Powell AJ, Pellagatti A, Lukashchuk N, McCance DJ, McCluggage WG, Schettino G, Salto-Tellez M, Boultwood J, Richard DJ, McDade SS, Harkin DP - Mol. Cell (2014)

BRCA1/BCLAF1-Mediated mRNA Splicing Is Required for Maintenance of ATRIP, BACH1, and EXO1 Protein Expression(A) Representative western blots demonstrating that depletion of either BRCA1 or BCLAF1 results in downregulated expression of ATRIP, BACH1, and EXO1 proteins in response to DNA damage.(B) Representative western blot demonstrating DNA damage-dependent accumulation of ATRIP, BACH1, and EXO1 proteins over time following inhibition of proteosomal mediated protein degradation with MG132 (10 μM). Cells were mock treated or treated with etoposide (1 μM) for 30 min prior to MG132 treatment.(C) Representative western blots demonstrating DNA damage-dependent increased protein turnover in control and BRCA1- or BCLAF1-depleted cells following inhibition of protein translation with Cyclohexamide (10 μg/mL). Cells were mock treated or treated with etoposide (1 μM) for 30 min prior to Cyclohexamide treatment.(D) Representative western blots demonstrating BRCA1 and BCLAF depletion in cells used for experiments shown in (C).(E–G) Quantification of ATRIP, BACH1, and EXO1 protein levels shown in (C). Image densitometry values were normalized to 0 hr and decay curves fitted and used to calculate protein half-lives.
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fig6: BRCA1/BCLAF1-Mediated mRNA Splicing Is Required for Maintenance of ATRIP, BACH1, and EXO1 Protein Expression(A) Representative western blots demonstrating that depletion of either BRCA1 or BCLAF1 results in downregulated expression of ATRIP, BACH1, and EXO1 proteins in response to DNA damage.(B) Representative western blot demonstrating DNA damage-dependent accumulation of ATRIP, BACH1, and EXO1 proteins over time following inhibition of proteosomal mediated protein degradation with MG132 (10 μM). Cells were mock treated or treated with etoposide (1 μM) for 30 min prior to MG132 treatment.(C) Representative western blots demonstrating DNA damage-dependent increased protein turnover in control and BRCA1- or BCLAF1-depleted cells following inhibition of protein translation with Cyclohexamide (10 μg/mL). Cells were mock treated or treated with etoposide (1 μM) for 30 min prior to Cyclohexamide treatment.(D) Representative western blots demonstrating BRCA1 and BCLAF depletion in cells used for experiments shown in (C).(E–G) Quantification of ATRIP, BACH1, and EXO1 protein levels shown in (C). Image densitometry values were normalized to 0 hr and decay curves fitted and used to calculate protein half-lives.
Mentions: As BRCA1 and BCLAF1 were required for efficient splicing and stability of spliced ATRIP, BACH1, and EXO1 transcripts following DNA damage, we next assessed the effect of BRCA1 or BCLAF1 depletion on ATRIP, BACH1, and EXO1 protein expression. In keeping with the reduced expression of ATRIP, BACH1, and EXO1 spliced transcripts, we observed substantially reduced expression of all three proteins in BRCA1- and BCLAF1-depleted cells following DNA damage (Figure 6A). However, to our surprise, in control cells we did not observe increased levels of ATRIP, BACH1, and EXO1 protein levels following DNA damage that would be expected, given the increased expression of spliced transcript observed. This suggests that either ATRIP, BACH1, and EXO1 translation is attenuated following DNA damage or or that turnover of these proteins may be increased and that BRCA1/BCLAF1-regulated cotranscriptional splicing serves to maintain the expression of these proteins in response to DNA damage.

Bottom Line: Mutations within BRCA1 predispose carriers to a high risk of breast and ovarian cancers.Here, we report the identification of a DNA damage-induced BRCA1 protein complex containing BCLAF1 and other key components of the mRNA-splicing machinery.Interestingly, mutations in a number of proteins found within this complex have been identified in numerous cancer types.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK. Electronic address: k.savage@qub.ac.uk.

Show MeSH
Related in: MedlinePlus